We propose a new approach for the selection of optimized gene-specific inhibitors based on libraries of self-circularizing antisense RNAs called Lassos. Lassos hybridize to and become topologically linked (throughcircularization) with target RNA molecules, creating complexes having superior stability and sequence specificitycompared to currently available antisense agents. This proposal addresses a major challenge in all antisense approaches, finding the most "sensitive" sequences on target RNAs. Our approach allows simultaneous selection of both accessible target sites and optimal circularizing ability for Lassos. The project includes the development of a new method for preparing gene-specific (directed)libraries and incorporating them into the Lasso structure. These directed libraries are of much lower complexity and toxicity than fully random libraries, making them especially useful for cell-based selections. A novel in vitro selection scheme will be used to identify Lasso species that bind and circularize around the target with fast kinetics, high affinity and sequence specificity. Phase II of the project will focus on applying the scheme developed in Phase I to selection in cells, to identify those Lassos that are best able to specifically inhibitexpression of a gene of interest. In this way, Lassos will be optimized for function in the cellular environment in an automatic and straightforward manner. They should find diverse applications, includinginhibitingthe expression of disease-related genes, functional genomics, and target validation.